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StemXVivo™ Procedure for the Ex Vivo Culture of CD3/CD28 Primed T Lymphocytes

Introduction

Hematopoietic cells are critical for the generation and regulation of immune responses, and dysfunction of these cells plays a key role in the etiology of many diseases. For this reason, maintenance of human T cell lines and clones in vitro is useful for the study of immune regulation. Serum-Free T Cell Base Media is a serum-free medium developed especially for the ex vivo expansion of T lymphocytes for research applications. Unlike traditional serum-containing culture media, serum-free medium provides a stable culture environment for the generation and maintenance of T cell lines and antigen-specific T cell clones. The medium is also applicable to routine culture of T cell lines and T cell clones, stimulation of PBLs, and study of mixed leukocyte reactions (MLR). A sample protocol for the culture of CD3/CD28 primed T cells is outlined below.

Reagents & Materials

  • Human StemXVivo Serum-Free T Cell Base Media (R&D Systems, Catalog # CCM010)
  • Human CD3+ T lymphocytes [isolated from peripheral blood mononuclear cells using the MagCellect Human CD3+ T Cell Isolation Kit (R&D Systems, Catalog # MAGH101), or Cell Enrichment Column (R&D Systems, Catalog # HTCC) or equivalent]
  • Antibodies:
    • Human CD3 monoclonal antibody (R&D Systems, Catalog # MAB100)
    • Human CD28 polyclonal antibody (R&D Systems, Catalog # AF-342-PB)
  • Penicillin-Streptomycin (Sigma, Catalog # P4333)
  • Recombinant human IL-2 (R&D Systems, Catalog # 202-IL, optional)
  • Tissue culture plates:
    • 96 well v- or round-bottom microplate (Corning Costar, Catalog # 3894, 3799, or equivalent)
  • 15 mL centrifuge tubes (Corning Costar, Catalog # 430052 or equivalent)
  • Serological pipettes
  • Pipettes and pipette tips
  • Hemacytometer
  • Centrifuge
  • Vortex mixer
  • Inverted microscope
  • 37° C, 5% CO2 incubator
  • [3H]-thymidine
  • Cell harvester
  • beta-scintillation counter

Procedure

  1. Prepare the CD3/CD28 coated 96 well microplate:
    • In a 15 mL conical tube, prepare a working solution of 1 µg/mL CD3 antibody and 1 µg/mL CD28 antibody in PBS.
    • Add 100 µL into each well of the 96 well microplate. Cover the plate and incubate for 90 minutes at 37° C or at 2 - 8° C overnight.
    • Remove the unbound antibodies from the plate by washing the well with 200 µL PBS three times prior to the addition of cells.
  2. Thaw the needed volume of the Human StemXVivo Serum-Free T Cell Base Media at 2 - 8° C or room temperature. Thawed media should be stable for up to 1 month in the dark at 2 - 8° C. Thoroughly mix the thawed medium by vortexing and warm the medium in a 37° C incubator (or water bath). If desired, antibiotics can be added to the medium at this point.
    Note: When handling biohazard materials such as human blood, universal precautions should be followed and protective clothing should be worn. For the handling of radioactive materials, safe laboratory procedures should be observed.

  3. Prepare CD3+ T cells from peripheral blood mononuclear cells according to the instructions in the MagCellect CD3+ T Cell Isolation Kit or Cell Enrichment Column.
  4. Resuspend the purified CD3+ T cells in Human StemXVivo Serum-Free T Cell Base Media, count the cells, and adjust the cell density to 1 x 106 cells/mL.
  5. Add the CD3+ T cells prepared in steps 3 and 4 to the plate prepared in step 1. Cells are added to each well at 2 x 105 cells in 0.2 mL. Place the plate in a humidified 37° C, 5% CO2 incubator for 3 - 5 days for short-term stimulation.
  6. The proliferation of CD3/CD28 primed T cells can be assessed by adding [3H]-thymidine to each well during the last 18 hours of a 3 - 5 day period. Harvest cells using a cell harvester and measure the cpm (counts per minute) with a beta-scintillation counter.

Other Applications

  • Proliferation of PBLs in the presence of the pharmacological agents (PMA, ionomycin, and A23187), lectins (PHA and ConA), or toxins (Staphylococcus enterotoxin).
  • Expansion of antigen-specific or non-specific T cells and T cell lines.
  • Mixed lymphocyte reactions (MLR).

Results

Representative results obtained from culturing purified CD3+ T cells from peripheral mononuclear cells with the Human StemXVivo Serum-Free T Cell Base Media (R&D Systems, Catalog # CCM010) over a 5-day period (Figure 1).

Figure 1
Figure 1: Proliferative response of cultured CD3/CD28 primed T cells in CCM010. 1 x 105 purified CD3+ T cells were cultured in uncoated or CD3- and CD28-coated 96 well microplates for 5 days in CCM010 or in RPMI supplemented with 10% fetal bovine serum (RPMI/10% FBS). [3H]-thymidine was added for the final 18 hours. Cells were harvested and the incorporation of [3H]-thymidine was measured using a beta-scintillation counter. Results are presented as the mean cpm of triplicate wells.

References

  1. Current Protocols in Immunology.
  2. Levine, B.L. et al. (1997) J. Immunol. 159:5921.
  3. Yamada-ohnishi, Y. et al. (2004) Stem Cells and Development 13:315.
  4. Levine, B.L. et al. (1996) Science 272:1939.
  5. Levine, B.L. et al. (1995) Int. Immunol. 7:891.
  6. King, C.L. et al. (1995) Eur. J. Immunol. 25:587.